Association of newly synthesized islet prohormones with intracellular membranes.

Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3H]tryptophan and [14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation. Extracts of supernatants and pellets were chromatographed by gel filtration; fractions were collected and counted. A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG. In contrast, glucagon- and somatostatin-related peptides exhibited much less membrane-association in SG (24-31%). Bacitracin, bovine serum albumin EDTA, RNAse, alpha-methylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone association with membranes. However, high salt (1 M KCl) significantly reduced membrane-association of prohormones. Binding of labeled prohormones to SG membranes from unlabeled tissue increased with incubation time and was inhibited by unlabeled prohormones. The pH optimum for prohormone binding to both M and SG membranes was 5.2. It is suggested that association of newly synthesized prohormones with intracellular membranes could be related to the facilitation of proteolytic processing of prohormones and/or transport from their site of synthesis to the secretory granules.

Benzoyl peroxide increases UVA-induced plasma membrane damage and lipid oxidation in murine leukemia L1210 cells.

Ultraviolet A radiation induces oxidative stress and cell damage. The purpose of this investigation was to examine whether ultraviolet A-induced cell injury was amplified by the presence of a non-ultraviolet A absorbing molecule capable of generating free radicals. Benzoyl peroxide was used as a lipid soluble potential radical-generating agent. Plasma membrane permeability assessed by trypan blue uptake was used to measure cell damage in murine leukemia L1210 cells. Cells were irradiated with a pulsed Nd/YAG laser at 355 nm using 0-160 J per cm2. The ratio of the fluence-response slope in the presence of 40 microM benzoyl peroxide to that of irradiated controls was 4.3 +/- 2.6. Benzoyl peroxide alone or benzoyl peroxide added after irradiation did not cause increased trypan blue uptake. The ratio of the fluence-response slopes in the presence of 40 microM benzoyl peroxide to that of irradiated controls was 4.7 +/- 1.4 when cells were irradiated (0-43 J per cm2) with a xenon lamp, filtered to remove wavelengths <320 nm. The increased trypan blue uptake in 355 nm-irradiated cells in the presence of benzoyl peroxide was inhibited in a concentration-dependent manner by butylated hydroxytoluene, vitamin E, and trolox, a water-soluble vitamin E derivative. Lipid oxidation, assessed as thiobarbituric acid reactive substances, was significantly increased in samples irradiated with ultraviolet A in the presence of benzoyl peroxide at fluences >34 J per cm2. The increased trypan blue uptake and thiobarbituric acid reactive substances were inhibited by butylated hydroxytoluene. These results suggest that agents not absorbing ultraviolet A radiation may enhance ultraviolet A-initiated oxidative stress in cells.

The effects of radicals compared with UVB as initiating species for the induction of chronic cutaneous photodamage.

There is substantial evidence that ultraviolet radiation induces the formation of reactive oxygen species which are implicated as toxic intermediates in the pathogenesis of photoaging. The aim of this study was to determine whether repeated topical treatment with benzoyl peroxide, a source of free radicals, produced the same cutaneous effects as chronic ultraviolet B radiation. Three concentrations of benzoyl peroxide (0.1, 1.5, 5.0% wt/wt) and three cumulative fluences of ultraviolet B radiation (0.9, 2.2, 5.1 J per cm2) used alone and in all combinations along with appropriate controls. Female SKH1 (hr/hr) albino hairless mice were treated 5 d per wk for 12 wk. Extracellular matrix molecules and histologic parameters were assessed. Ultraviolet B radiation induced a fluence-dependent and time-dependent increase in skin-fold thickness. Fluence dependence was seen for epidermal thickness, sunburn cell numbers, dermal thickness, glycosaminoglycan content, mast cell numbers, and skin-fold thickness. Benzoyl peroxide treatment alone caused less marked increases in epidermal and dermal measures compared with ultraviolet B under the conditions used. A benzoyl peroxide concentration-dependent increase was only observed for elastin content, although the highest concentration of benzoyl peroxide increased epidermal thickness and glycosaminoglycan content. A synergistic interaction between ultraviolet B and benzoyl peroxide was not found. These results indicate that repeated administration of benzoyl peroxide produces skin changes in the hairless mouse that qualitatively resemble those produced by ultraviolet B and suggest that common mechanisms may be involved. In addition, any potential synergistic effect of ultraviolet B and benzoyl peroxide was below the level of detection used in this study.

NMR analyses of the cold cataract. III. 13C acrylamide studies.

13C-enriched acrylamide was employed to further delineate the action of this compound in preventing the cold cataract phenomenon when it is incorporated (in vitro) into young human and rabbit lenses. The extent of acrylamide incorporation, in the dark and with concurrent UV exposure, was monitored by 13C NMR spectroscopy. These studies provide further evidence that UV exposure causes permanent acrylamide photobinding within the lens. In such lenses, the gamma crystallin fraction of the soluble lens proteins is affected to the greatest extent. It appears to become aggregated and/or combined with the alpha and beta fractions resulting in an apparent loss of most of the gamma monomers. There is also an age-related effect with respect to the amount of acrylamide that can be incorporated into the lens. The decrease in acrylamide incorporation with age directly parallels the age-related decline in gamma crystallin levels.

Further studies on the effects of UV radiation on the human lens.

Young (1st decade) and old (7th decade) normal human lenses were exposed to low-level (less than 1 mW/cm2) broadband UV radiation (300-400 nm). UV-induced effects in the whole lens and the extracted lens proteins were monitored by fluorescence spectroscopy. The lens proteins were also subjected to proton and pulse magnetization NMR spectroscopy. These studies provide further evidence that low-level UV radiation exposure enhances nontryptophan fluorescence and may generate additional (longer wavelength) chromophores. The NMR studies suggest that the young gamma crystallin fraction is particularly sensitive to UV radiation compared with the other crystallins.